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Research Article | | Peer-Reviewed

Histopathological Effects of the Entomopathogenic Fungus Metarhizium anisopliae (Metschnikoff, Sorokin) on Adults of Rastrococcus invadens, (Williams, 1986)

Fawrou Seye*,  Mamour Toure,  Amadou Fall,  Fatou Kamby,  Mady Ndiaye
Published in American Journal of BioScience (Volume 13, Issue 4)
Received: 27 July 2025     Accepted: 7 August 2025     Published: 26 August 2025
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Abstract

The mealybug (Rastrococcus invadens), is a major pest of mango trees, causes extensive damage and significant yield losses in Senegal. The control methods used seem less effective. Mechanical control is the most widely used method in some regions of Senegal. However, the use of natural products has shown promising potential. The aim of this study was to investigate the pathogenicity of Metarahizium anisoliae on adults mealybugs (Rastrococcus invadens) and to assess the resulting histological damage. Under laboratory conditions (75% relative humidity and 27°C), we tested oil-based formulations of Metarhizium anisopliae conidia at doses of 103, 104, 105, 106, and 107 spores/ml to mango leaf discs infested with adult Rastrococcus invadens. Treated insects were examined using optical microscopy with histological techniques to observe tissue damage. The results revealed that mortality rates were higher 48 hours after application, with respective rates of 37.4±8.8%, 58.6±5.22%, 75.45±5.28%, 92.35±1.69%, and 100±0.01% compared to the control group at 2.4±1.2%. Histological sections showed a progressive invasion of fungal spores through the cuticle, leading to the destruction of fatty tissues, muscles, and wax glands, followed by internal proliferation. These findings suggest that entomopathogenic fungi such as M. anisopliae can be used and popularized as biopesticides in the management of R. invadens.

Published in American Journal of BioScience (Volume 13, Issue 4)
DOI 10.11648/j.ajbio.20251304.11
Page(s) 96-101
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This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

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Copyright © The Author(s), 2025. Published by Science Publishing Group
Previous article
Keywords

Rastrococcus Invadens, Histopathology, Metarhizium Anisopliae

1. Introduction
Rastrococcus invadens was first identified in Dakar, Senegal in 1995
[1]
. Since then, it has spread widely, causing damage to fruit trees, particularly mango trees, in regions with high production, especially Thiès and Casamance
[2]
. R. invadens is a polyphagous, bisexual species that attacks many citrus and mango species
[3]
, and its female has a pre-reproductive period, producing up to 200 larvae in its lifetime
[4]
. Recent studies have shown morphometric
[5]
and genetic
[6]
characterization of R. invadens in these regions. Control methods practiced by producers are generally ineffective in controlling R. invadens infestation. Recent studies have shown the possibility of controlling this insect using parasitoids
[7]
or biopesticides such as plant extracts
[8-10]
or entomopathogenic fungi
[11, 12]
. Among entomopathogenic fungi, M. anisopliae is widely used against various aquatic
[13, 14]
and terrestrial insect species
[15-17]
. The pathogenicity of M. anisopliae strains has been demonstrated in various studies
[18]
. In most studies, it has been shown that the fungus acts through the insect cuticle
[19]
. Given the pathogenicity of M. anisopliae demonstrated in recent studies on R. invadens
[11]
, we deemed it necessary to evaluate a local strain of M. anisopliae on R. invadens and show histological damage.
2. Materials and Methods
2.1. Animal Material
Given that male Rastrococcus invadens are winged, only females were used, which are fixed on mango leaves. They were sampled from mango leaves of the "Sewe" variety in the garden of the Baccalaureate Office of Cheikh Anta Diop University of Dakar (latitude 14° 41'16" N, longitude 17°28'0") and transported directly to the laboratory for testing.
2.2. Plant Material
The Metarhizium (Metchnikoff Sorokin) strain was provided by the Plant Protection Directorate (DPV) in Dakar. The lyophilized spores in powder form were stored in aluminum packets under laboratory conditions and kept refrigerated (4°C). Vegetable oil was used to formulate the spore oil suspension. Different concentrations were adjusted using a Malassez cell.
2.3. Toxicity Test
The applied doses were 103, 104, 105, 106, and 107 spores/ml. Spraying was done using ALFA 500 manual sprayer for horticultural sprayer directly on insects fixed on freshly harvested mango leaves. Vegetable oil was used only as a control. Mortality was assessed 24 and 48 hours after spraying. Mortality rates were corrected using Abbott's formula
[20]
. Three repetitions were performed for each test, and means were reported.
2.4. Histological Study
To show histological damage, treated adults were fixed with the highest dose that caused 100% mortality and those of the control group after 48 hours. Insects were randomly selected from the treated group and fixed in a Carnoy solution (60% ethanol + 30% chloroform + 10% acetic acid). The technique used to perform sections on Rastrococcus invadens adults was standardized and described
[21]
. Staining was done with Hematoxylin Eosin (HE). Optical microscope “Optika” was used with an integrated camera. Images were captured on a computer and recorded in JPEG format.
3. Results
3.1. Toxicity
At 24 hours, mortality was observed for all doses. However, mortality rates were only high 48 hours after application (Figure 1), with successive rates of 37.4±8.8%, 58.6±5.22%, 75.45±5.28%, 92.35±1.69%, and 100±0.01% compared to the control group at 2.4±1.2%.
Figure 1. Mortality of Rastrococcus invadens adults treated with a Metarhizium anisopliae oil solution (C1=103, C2=104, C3=105, C4=106, and C5=107 spores/ml) 24 and 48 hours after treatment. Values are mean corrected percent mortality of four replicates with standard error (SE).
3.2. Histopathological Effects
Histological examination reveals a degradation of internal tissues. A comparison with the control sample shows significant disintegration of internal tissues in the treated samples. At the level of the overall view, we observe in longitudinal section a degradation of internal tissues of R. invadens within 24 hours (Figure 2b) compared to the control (Figure 2a).
Figure 2. Longitudinal sections of Rastrococcus invadens adults untreated (figure 2a) and treated with Metarhizium anisoplaie oil formulated (Figure 2b) 48 hours after treatment (X40). m= muscle, fb= fat body, cu= cuticle (X40), coloration HE.
Figure 3. Histological section of the peripheral zone (cuticle and fat body) of untreated Rastrococcus invadens adults (Figure 3a, 3b) and treated with a Metarhizium anisopliae oil suspention (Figure 3c and 3d) 48 hours after. Cer. gl= wax gland, fb= fat body, cut= cuticle, con. set= conical setae, coni.= conidia (X400).
At high magnification (X400), we observe destruction of the wax glands near the cuticle of treated R. invadens adults (figure 3c) compared to the intact glands of untreated adults (figure 3b). Mycelium develops through the cuticle (Figure 3c) and invades the adipose tissue (Figure 3c and 3d).
Figure 4. Histological section of untreated Rastrococcus invadens adult (Figure 4a) and treated (Figure 4b, 4c, 4d) with Metarhizium anisopliae oil formulation 48 hours after. tm = transversal muscle, lm= longitudinal muscle, myc = mycelium (X400).
Compared to the control (Figure 4a), we observe at high magnification a development of mycelium in the muscle tissue in many areas (Figure 4b, 4c, 4d). This development is accompanied by massive and almost complete destruction of the muscle tissue in treated adults. Microscopic examination shows fungal growth in the insect's internal cavity, leading to disintegration of internal tissues. The fungus colonizes the internal cavity, causing extensive tissue damage and disruption of vital organs, ultimately contributing to the insect's mortality.
4. Discussion
The application of the Metarhizium anisopliae oil formulation showed increasing mortality rates of 37.4±8.8%, 58.6±5.22%, 75.45±5.28%, 92.35±1.69%, and 100±0.01% for doses ranging from 103 to 107 compared to the control group at 2.4±1.2% within 48 hours. The formulation of spores with vegetable oil seems to facilitate adhesion of conidia and the infestation time. This leading to high mortality rates within 48 hours compared to other study
[18]
. Indeed, these authors observed mortality rates between 48 and 96 hours after applying M. anisopliae spores in aqueous suspension. This suggests that spores adhere more easily in large numbers with an oil formulation than with an aqueous formulation. In fact, our previous work has shown that oil formulations of entomopathogenic fungi can increase the effectiveness of spores on insects
[14, 22, 23]
. However, in this study, we did not evaluate the germination capacity of spores on the insect beyond 48 hours.
However, through optical microscopy, the sections performed showed that M. anisopliae spores caused destruction of internal structures of R. invadens. The spores were able to penetrate through the cuticle before developing in the hemocoel and fat body. Spore development was also observed in muscle tissue and exocrine glands such as wax glands. These microscopic observations demonstrate a mechanism of action of M. anisopliae on insects, confirming several studes
[16, 24]
. In fact, studies have shown that after penetration through the cuticle or after ingestion, the spores begin their development by excreting secondary metabolites, particularly toxins that disorganize and destroy the surrounding tissues
[24, 25]
. Since R. invadens is a sap-feeding insect, the spores could only penetrate through the cuticle and not through the digestive tract.
This led us to focus on tissues other than the digestive tract, unlike other insects that feed only by ingestion, such as mosquito larvae
[21, 24, 25]
or locusts
[26]
.
Furthermore, we were unable to identify and examine other tissues such as those of the digestive system, nervous system, and reproductive organs. However, the proliferation of mycelium in the internal structures of R. invadens would likely not leave other internal tissues intact.
5. Conclusion
This study highlights the importance of Metarhizium anisopliae oil formulated againts pests as Rastrococus invadens. The vegetable oil enhances the effectiveness of the fungus by facilitating the adhesion of conidia that penetrate through the cuticle before destroying internal tissues. further histological studies on the effects of the fungus on the reproduction or biology of R. invadens is necessary to assess its pathogenicity on development.
Abbreviations

HE

Hematoxylin Eosin

DPV

Plant Protection Directorate

SE

Standard Error
Author Contributions
Fawrou Seye: Conceptualization, Methodology, Writing - original draft
Mamour Toure: Writing - original draft
Amadou Fall: Conceptualization
Fatou Kamby: Methodology
Mady Ndiaye: Supervision
Funding
This work is not supported by any external funding.
Conflicts of Interest
The authors declare no conflicts of interest.
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    Seye, F., Toure, M., Fall, A., Kamby, F., Ndiaye, M. (2025). Histopathological Effects of the Entomopathogenic Fungus Metarhizium anisopliae (Metschnikoff, Sorokin) on Adults of Rastrococcus invadens, (Williams, 1986). American Journal of BioScience, 13(4), 96-101. https://doi.org/10.11648/j.ajbio.20251304.11

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    Seye, F.; Toure, M.; Fall, A.; Kamby, F.; Ndiaye, M. Histopathological Effects of the Entomopathogenic Fungus Metarhizium anisopliae (Metschnikoff, Sorokin) on Adults of Rastrococcus invadens, (Williams, 1986). Am. J. BioScience 2025, 13(4), 96-101. doi: 10.11648/j.ajbio.20251304.11

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    AMA Style

    Seye F, Toure M, Fall A, Kamby F, Ndiaye M. Histopathological Effects of the Entomopathogenic Fungus Metarhizium anisopliae (Metschnikoff, Sorokin) on Adults of Rastrococcus invadens, (Williams, 1986). Am J BioScience. 2025;13(4):96-101. doi: 10.11648/j.ajbio.20251304.11

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  • @article{10.11648/j.ajbio.20251304.11,
  author = {Fawrou Seye and Mamour Toure and Amadou Fall and Fatou Kamby and Mady Ndiaye},
  title = {Histopathological Effects of the Entomopathogenic Fungus Metarhizium anisopliae (Metschnikoff, Sorokin) on Adults of Rastrococcus invadens, (Williams, 1986)
},
  journal = {American Journal of BioScience},
  volume = {13},
  number = {4},
  pages = {96-101},
  doi = {10.11648/j.ajbio.20251304.11},
  url = {https://doi.org/10.11648/j.ajbio.20251304.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajbio.20251304.11},
  abstract = {The mealybug (Rastrococcus invadens), is a major pest of mango trees, causes extensive damage and significant yield losses in Senegal. The control methods used seem less effective. Mechanical control is the most widely used method in some regions of Senegal. However, the use of natural products has shown promising potential. The aim of this study was to investigate the pathogenicity of Metarahizium anisoliae on adults mealybugs (Rastrococcus invadens) and to assess the resulting histological damage. Under laboratory conditions (75% relative humidity and 27°C), we tested oil-based formulations of Metarhizium anisopliae conidia at doses of 103, 104, 105, 106, and 107 spores/ml to mango leaf discs infested with adult Rastrococcus invadens. Treated insects were examined using optical microscopy with histological techniques to observe tissue damage. The results revealed that mortality rates were higher 48 hours after application, with respective rates of 37.4±8.8%, 58.6±5.22%, 75.45±5.28%, 92.35±1.69%, and 100±0.01% compared to the control group at 2.4±1.2%. Histological sections showed a progressive invasion of fungal spores through the cuticle, leading to the destruction of fatty tissues, muscles, and wax glands, followed by internal proliferation. These findings suggest that entomopathogenic fungi such as M. anisopliae can be used and popularized as biopesticides in the management of R. invadens.},
 year = {2025}
}

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  • TY  - JOUR
T1  - Histopathological Effects of the Entomopathogenic Fungus Metarhizium anisopliae (Metschnikoff, Sorokin) on Adults of Rastrococcus invadens, (Williams, 1986)

AU  - Fawrou Seye
AU  - Mamour Toure
AU  - Amadou Fall
AU  - Fatou Kamby
AU  - Mady Ndiaye
Y1  - 2025/08/26
PY  - 2025
N1  - https://doi.org/10.11648/j.ajbio.20251304.11
DO  - 10.11648/j.ajbio.20251304.11
T2  - American Journal of BioScience
JF  - American Journal of BioScience
JO  - American Journal of BioScience
SP  - 96
EP  - 101
PB  - Science Publishing Group
SN  - 2330-0167
UR  - https://doi.org/10.11648/j.ajbio.20251304.11
AB  - The mealybug (Rastrococcus invadens), is a major pest of mango trees, causes extensive damage and significant yield losses in Senegal. The control methods used seem less effective. Mechanical control is the most widely used method in some regions of Senegal. However, the use of natural products has shown promising potential. The aim of this study was to investigate the pathogenicity of Metarahizium anisoliae on adults mealybugs (Rastrococcus invadens) and to assess the resulting histological damage. Under laboratory conditions (75% relative humidity and 27°C), we tested oil-based formulations of Metarhizium anisopliae conidia at doses of 103, 104, 105, 106, and 107 spores/ml to mango leaf discs infested with adult Rastrococcus invadens. Treated insects were examined using optical microscopy with histological techniques to observe tissue damage. The results revealed that mortality rates were higher 48 hours after application, with respective rates of 37.4±8.8%, 58.6±5.22%, 75.45±5.28%, 92.35±1.69%, and 100±0.01% compared to the control group at 2.4±1.2%. Histological sections showed a progressive invasion of fungal spores through the cuticle, leading to the destruction of fatty tissues, muscles, and wax glands, followed by internal proliferation. These findings suggest that entomopathogenic fungi such as M. anisopliae can be used and popularized as biopesticides in the management of R. invadens.
VL  - 13
IS  - 4
ER  -

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